Prime-boost vaccination using recombinant Mycobacterium bovis BCG and recombinant vaccinia virus DIs harboring HIV-1 CRF01_AE gag in mice: influence of immunization routes.

We have previously reported that live vector-based HIV-1 gag vaccine candidate using BCG as a vector was achievable in BALB/c mice. Although the gag-specific CTL induced by this live candidate vaccine is significantly high, persistence of CTL remains unclear. Thus, efforts were made to explore the potential of recombinant Vaccinia virus DIs strain harboring the same HIV-1 CRF01_AE gag gene (rVaccinia/ HIV-1gagE) present in the BCG construct, using different immunization routes. After one month following a single subcutaneous (s.c.) injection of rBCG/HIV-1gagE, higher CTL responses were recognized against various peptide epitopes along the whole gag protein compared to that by intradermal (i.d.) route. A prime-boost regimen having only rDIs/HIV-1gagE injected i.d. induced very low CTL levels. However, within two months, by priming with rBCG/HIV-1gagE s.c. and boosting with rVaccinia/HIV-1gagE intravenously (i.v.), CTL levels were greater (20-68% specific cell lysis) than those obtained by priming and boosting both i.d. (18-35%). After seven months, both prime-boost immunization with rBCG/HIV-lgagE s.c. and with rVaccinia/HIV-1gagE either i.v. or i.d. sustained similar CTL levels. Our studies exhibit that the prime-boost vaccination of rBCG/HIV-1gagE following by rVaccinia/HIV-1gagE i.d. could be used to elicit prolonged CTL responses as well as memory T-cells in mice, which might be more practical than using i.v. route.

Enhancement of cell-mediated immune response in mice by whole HIV-1 gag in Mycobacterium bovis BCG as a live vaccine candidate.

In this study, we employed a recombinant Mycobacterium bovis Bacille Calmette-Guerin (BCG) harboring whole HIV-1 CRF01_AE gag DNA as a candidate vaccine to investigate specific cell-mediated immunity in BALB/c mice. Construction of the stable expression recombinant BCG was achieved by demonstrating by Western blot detection of protein of approximately 55 kDa. By a single injection of 0.1 mg of the recombinant HIV-1 gag protein expressing BCG subcutaneously into mice, after 2 weeks various specific cytotoxic T-lymphocyte (CTL) responses were exhibited against a single gag epitope of amino acid positions 294-304, and also against various peptide regions along the entire gag protein with moderate CTL activities (10-35% specific cell lysis), which increased to relatively high levels (50-68%) after one month. However, after two months activities were 3-3.7 fold lower. On the other hand, gag-specific lymphocyte proliferation was detected 9.3 fold higher than that of non-immunized mouse spleen cells. Our results indicate that in mice, BCG can be used as a recombinant live vector to induce cellular immune responses to HIV-1 gag antigen.

Identification of clinical isolates of Leptospira spp by pulsed field gel-electrophoresis and microscopic agglutination test.

Twenty-five clinical isolates of Leptospira spp were characterized by microscopic agglutination test (MAT) and pulsed field gel-electrophoresis (PFGE) in comparison with 23 reference Leptospira serovars and with the saprophytic L. biflexa serovar Patoc. PFGE DNA profiling was more specific and reliable than MAT.

Specific immune response and pathological findings in BALB/c mice inoculated with recombinant BCG expressing HIV-1 antigen.

Recombinant BCGs (rBCGs) containing extrachromosomal plasmids with different HIV-1 insert sequences: nef, env (V3J1 and E9Q), gag p17 or whole gag p55 were evaluated for their immunogenicity, safety and persistent infection in BALB/c mice. Animal injected with, rBCG-plJKV3J1, rBCG-pSO gag p17 or rBCG-pSO gag p55 could elicit lymphocyte proliferation as tested by specific HIV-1 peptides or protein antigen. Inoculation with various concentration of rBCG-pSO gag p55 generated satisfactory specific lymphocyte proliferation in dose escalation trials. The rBCG-pSO gag p55 recovered from spleen tissues at different time interval post-inoculation could express the HIV protein as determined by ELISA p24 antigen detection kit. This result indicated that the extrachromosomal plasmid was stable and capable to express Gag protein. It was also demonstrated that rBCGs did not cause serious pathological change in the inoculated animals. The present study suggested the role of BCG as a potential vehicle for using in HIV vaccine development.

Mortality analysis of HIV-1 infected patients for prioritizing antiretroviral drug therapy.

Mortality data of patients, classified according to their clinical status and CD4+ cell count status, would be very useful to guide clinicians to prioritizing patients who need antiretroviral drug therapy. In the current study, the authors re-analyzed data derived from a previously published retrospective study of HIV-1-infected individuals at Lampang Hospital in northern Thailand. According to the Cox proportional hazard model, compared to asymptomatic patients with a high CD4+ cell count (> 200 cell/microl), the mortality rate of asymptomatic patients with a medium CD4+ cell count (100-199 cell/microl) did not significantly differ. However, the mortality rate of patients with a CD4+ cell count below 100 cell/microl was at least 16 times higher, regardless of the presence of clinical symptoms. Based on these results, the authors produced a Lampang Hospital guideline of antiretroviral drug use; priority of antiretroviral therapy should, therefore, be given to patients with CD4+ cell count < 100 cell/microl.

Lymphocyte and NK cell subpopulations in HIV seronegative Thais.

Lymphocyte subpopulations, i.e. T, B and natural killer (NK) cells including NK cell subsets which express CD16 molecules (with or without co-expression of CD56 molecules) and NK cell subsets which express CD56 molecules (with or without co-expression of CD16 molecules) were enumerated by two color-flow cytometry in a total of 125 HIV seronegative Thai adults. The study demonstrated relatively low CD4 counts in the subjects, i.e. 26.3% of them had a CD4 count of less than 500 cells/microl. In contrast, their NK cell counts were relatively high. Statistical analyses of the percentage values showed that females had significantly higher CD3 (total T cells), but lower NK cell counts as compared to males (p < 0.05). Regarding age variation, an increase of 1.1% of CD4 cells per decade was seen. It was roughly estimated that about 86% of NK cells harbored both CD16 and CD56 molecules. Collective data from several studies including the present one suggest that high NK cell counts may be a compensation for low CD4 cell counts in Mongoloid people. Thus, the role of NK cells in the defense cascade against viral infections, especially human immunodeficiency virus infections deserves further investigation.

Characteristic of HIV-1 in V3 loop region based on seroreactivity and amino acid sequences in Thailand.

The third variable (V3) domain of the envelop (env) protein has been used for determining genetic subtype and phenotypic characteristics of human immunodeficiency virus type 1 (HIV-1) isolates. Based on the seroreactivity of the HIV-1 subtype by V3 peptide binding enzyme immunoassay (EIA) of 351 samples obtained in 1998 from HIV-1 infected individuals and AIDS patients, we found that 283 (80.6%) were subtype E, 20 (5.7%) were subtype B, 28 (8.0%) were cross-reactive between both types and 20 (5.7%) were non-typeable. The degree of seroreactivity of HIV-1 subtype E decreased significantly when the amino acid at the crown of the V3 loop was substituted from a GPGQ motif to GPGR motif. Interestingly, AIDS patients who had V3 sequences of subtype E as GPGR motif had a stronger immunoreactivity to GPGQ motif peptides than to GPGR motif peptides, in contradiction for their proviral sequences. The results suggested that mutations in the V3 loop may lead to a changed immunoreactivity that makes HIV-1 mutants unrecognizable or allow escape from the primary immune response by means of neutralizing sensitivity. In connection with vaccine development, it should be pointed out that the combination of V3 sequencing and peptide EIA could provide a novel approach to obtain a primarily infected virus sequence as a target for a preventive AIDS vaccine.